A rapid explosion in the sequencing of entire genomes has increased the need for highly parallel methods that allow simultaneous investigation of several thousands of genes in a highly miniaturized fashion. Parallel study of thousands of genes at the genomic level promises to be a critical element in understanding and curing disease. For this reason, among others, high-throughput analysis methods are imperative to the future of medicine including gene discovery, disease diagnosis, genotyping, protein expression, elucidating metabolic responses, drug design, drug discovery and toxicology.
One such tool capable of investigating several thousands of molecules in parallel is an array (Shi, 2002). Briefly, an array is an ordered arrangement of compounds, including biological and biochemical materials, and serves as a medium for matching samples based on complementarity or selective chemical reactions. A microarray is a specific array and is distinguished by samples sizes of less than 200 microns in diameter. The microarray is a device comprising several molecules or more biomolecules of known identity, attached to or immobilized on a surface of a substrate or solid support. The molecules or biomolecules are applied iteratively to the substrate in a highly parallel fashion to generate a discrete spatial grid such that an array having elements corresponding to particular complementary molecules or biomolecules is produced. Generally, the attached or immobilized molecules are the “probe”, and the complementary species is the “target”. The “target is typically the analyte or species to be detected or quantitated. Nevertheless, in some applications these roles may be reversed and the target may be immobilized while the probe may be free. In the general case, the probe is a molecule to be analyzed (i.e., the “analyte”) which is often of unknown identity and, in some cases, is extracted from a sample of interest and labeled, such as with a fluorescent dye, for ready detection. The labeled target(s) is incubated with the microarray under hybridizing conditions and allowed to bind to its complementary probe on the array. After removing the unbound probe, the amount of bound probe is detected and quantitated.
Reliance on microarrays for biochemical investigations has increased because of their demonstrated high throughput capacity. Various methods of microarray manufacture, use and improvements thereon have been described. For example, U.S. Pat. No. 5,744,305 and U.S. Pat. No. 5,445,934 to Foder et al. teach methods of synthesizing polymers, particularly oligonucleotides, polynucleotides and peptides, in an array format on a planar, non-porous solid support. The synthetic regions are prepared and defined by lithography methods that involve passing light through a mask to activate the exposed region for synthesis of the polymer. The resulting derivatized substrate, or array device, comprises polymers attached to the surface of the support in the regions activated by the light treatment.
U.S. Pat. No. 5,807,522 to Brown et al. teaches a spotting method of fabricating microarrays for biological samples in which a solid support having a discrete sample-analysis region prepared by applying a selected, analyte-specific reagent to the solid support using an elongate capillary channel and a tip region at which the solution in the channel forms a meniscus, tapping the tip of the dispensing device against the solid support at a defined position on the surface, with an impulse effective to break the meniscus in the capillary channel and depositing a selected volume between 0.002 and 2 nl of solution on the surface. Iterative steps of depositing the analyte-specific reagent to the solid support produces the final microarray. Brown et al. also teaches that the solid support comprises a substrate having a water impermeable backing, and atop the backing is a water permeable film formed of a porous or a non-porous material that is, for example, in a grid that is formed by applying a barrier material, such as silicon, by mechanical pressure or printing to form a water-proof barrier separating regions of the solid support.
U.S. Pat. No. 6,210,894 teaches that surrounding each array element, which is hydrophilic, with hydrophobic regions prevents potential cross-contamination caused by spreading of solution that are spotted on the surface of the microarray. This modified surface characteristic establishes clear boundaries between array elements. Further, it was suggested that the drop of the solution is deposited in such a manner as to synchronize spatially with each of the hydrophilic array elements, however, requiring alignment imposes a substantial restriction on the dispensing equipment, which, in many cases, is difficult to attain with conventional devices.
WO 01/73126 to Lyles describes a diagnostic device comprising a matrix having fibers of silica, alumina or their combination to provide a rigid, three-dimensionally continuous network of open, intercommunicating voids. The structure is taught to be readily modified by chemical reactions for binding a compound with increased loading capacity (see also U.S. Pat. No. 5,951,295 to Lyles). U.S. Pat. No. 5,629,186 to Yasukawa et al. also teaches rigid fused silica, alumina, or silica and alumina fiber matrices, and the matrices are useful for as a body implantable material, for supporting tissue growth in vivo, for in affinity chromatography and for blood diagnostic assays.
U.S. Pat. Nos. 5,700,637 and 6,054,270 to Southern teach arrays prepared by methods involving iterative coupling of a nucleotide precursor to form the array. The surface is taught to include a smooth, impermeable surface such as glass, and a surface having sintered microporous glass placed in microscopic patches.
WO 99/32663 teaches a system for detecting a molecule in a sample comprising a substrate having multiple spatially discrete regions, wherein the region comprises at least eight different anchors, each in association with a bifunctional linker, and at least two regions are identical. The regions on the surface may comprise a subregion for purposes of reducing the tolerance required for physically placing a single anchor (or group of anchors) into each designated space, and providing uniformity to the size of the areas containing the anchors, thereby facilitating the detection of the molecule.
Two main ways of preparing a microarray using flat plain glass as substrates have been described—light directed in-situ synthesis of a probe, and immobilization of synthesized biomolecules onto solid substrates that serve as probes for the microarray (WO 90/03382; WO 93/22680; U.S. Pat. No. 5,412,087 to McGall et al.; WO 95/15970). Methods in the prior art to increase the surface area of a microarray, and consequently the throughput capacity and sensitivity, have involved, for example, preparing a porous substrate, wherein the pores serve a sites for attachment of a plurality of biomolecules. For example, Nagasawa et al. (U.S. published application 2001/0039072) teaches a reactive probe chip comprising a composite substrate having compartments (i.e., wells) within which loaded porous carrier particulate probes are immobilized. Nagasawa et al. teaches that it is critical that the immobilization of the carrier particulate probes occur only at the outer surfaces and protective measures, such as impregnating with water, are taught to prevent damage to the inner pore surface, which carries the bound probe, during immobilization. Another approach to improving low probe density is described in WO 00/61282, which teaches a porous substrate for making a microarray that has two regions, a support region and a porous region. This porous region is described as offering substantial advantages over flat glass, and porous regions created by depositing a thin film comprising colloidal silica were found to improve signal enhancement 15 to 45 times that of flat glass, wherein the thicker films and smaller particles afforded that better results. However, accessiblity of biomolecules in the optimal systems was not maximized, i.e., the means for molecules to penetrate the pores is difficult and kinetically challenging with respect to achieving sufficient proximity to a specific binding site. Further, in the wash step, this problem manifests itself because non-specific molecules get physically trapped within the pores.
PCT application WO 01/61042 teaches smooth surfaced porous membranes having one or more advantages such as low autofluorescence, thermal-cyclability, especially under humid conditions, and three-dimensional binding capacity. The membrane is a composite membrane comprising a porous polymer layer disposed on a support, which are disclosed for the use of making a microarray device, and the porous layer is characterized by having specific surface characteristics (e.g., surface roughness).
The problem of accessibility in designing high surface area microarrays is observed in WO 01/16376, which teaches a substrate for the attachment of an array of biomolecules comprising a substantially planar, rigid inorganic material having a top surface, wherein the top surface has a plurality of pores disposed therein. Further, the planar, top surface further comprises a cationic polymer, such as polylysine, bonded to the surface to afford ready attachment of negatively charged biomolecules, including polynucleotides. In describing suitable inorganic materials for providing the pores within the top surface of the substrate, it was observed that sol-gel coating and Vycor provided relatively poor accessibility of the polynucleotide (i.e., DNA) for hybridization.
The porous coatings described in the prior art resulted in at least one of several problems such as inadvertent trapping of the targets (and probes), poor access of longer and/or larger molecules to the pores and long hybridization times. The inadvertent trapping of the molecules result in the background signal to rise and thus negate the advances made in the increased signal. One attempt to overcome the problems associated with poor molecular accessibility is described in U.S. Pat. No. 5,843,767, which teaches creating capillary tubes that bind an analyte to a surface of the tube and function to carry fluids comprising probes. Thus, the probes are accessible to the analytes. However, the construction introduces many constraints in the chip design, and the analysis requires special equipment, thereby adding to the cost.
These results indicate that effecting a high surface area to a microarray device involves not only a quantitative increase in the surface area, such as with disposing pores on a planar top surface, but also a consideration of accessibility the surface area to the target and/or probe. Otherwise, the structural increase in surface area does not fully translate into a microarray device having increased surface area.
The present invention alleviates these problems in the prior art by providing systems and methods directed to a substrate having a high surface area in each of three dimensions for making a microarray that realizes the full potential of the surface area. The invention is well-suited in the fabrication of microarray devices that are employed in biological and chemical analyses of biomolecules of various sizes and dimensions. Specifically, the substrate comprises surface micro-features that are smaller than each of the array elements. These micro-features have specific surface characteristics and texture, such as a tailored porosity, that is useful for the analysis of chemicals, particularly biomolecules.